This invention relates to a process and a kit including materials for diagnosing disease and particularly relates to a process for differentially diagnosing various rheumatological diseases which are characterized by the presence of autoimmune components.
Autoimmune diseases may be classified into two broad categories: systemic and organ-specific diseases. Within the systemic category, a group of disorders known as rheumatic diseases have been identified. This group includes systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and mixed connective tissue disease (MCTD). Because of the systemic nature of these diseases, patients suffering from any of the three diseases tend to present the same clinical symptoms at least during the initial stages; distinguishing among these diseases is, therefore, often quite difficult. An evaluative test which would provide for early diagnosis and discrimination of SLE, RA and MCTD would be clearly useful.
Historically, the diagnosis of SLE included, among other criteria, the identification of the lupus erythematosus (LE) cell. This cell, a polymorphonuclear leukocyte, is characterized by the presence of a large amorphous inclusion generally believed to be comprised of the nuclear contents of yet another of the patient's own white blood cells. A test based on this criterion is far from definitive in that although close to 80% of SLE patients possess the LE cell, over 70% of people who are "LE cell positive" do not have SLE.
The advent of immunofluorescent techniques allowed for the identification of a group of autoantibodies which were regularly associated with SLE. These autobodies had as their target the nuclei of the patient's own cells and became known as antinuclear antibodies (ANA). Unfortunately, even though 100% of the SLE patients displayed ANA so did 100% of R.A. and M.C.T.D. patients.
This non-specificity is not surprising when one considers the wide variety of antigenic targets which exist in the nucleus. In SLE alone, autoantibodies against such nuclear components as nucleo-protein, nuclear glycoprotein, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), polynucleotides, and a group of antigens known as extractable nuclear antigens (ENA) have been identified.
Although the molecular identities of some of the nuclear antigens are known (e.g. DNA:(Adams, L. E. et al. Amer. J. of Clin. Patn. 77(1):54-59 (1982)), RNA:(Lerner M. R. et al. Science Vol. 211:400-402 (1981)) and histones:(Aitkaci, A. et al J. Imm. Meth. 44:311-322 (1981)), the majority are defined only by their immunological or physiochemical characteristics. The identification of these antigens as proteins with specific cellular functions could lead to the elucidation of the etiologies of the autoimmune diseases as well as a method of differential diagnosis of the diseases.
Currently, a majority of the diagnostic tests for rheumatological diseases such as SLE are based upon identification of autoantibodies to DNA. For example:
U.S. Pat. No. 4,234,563 to D. R. Rippe Nov. 18, 1980 discloses a method detecting antibodies in SLE patents which are directed to DNA. The DNA is conjugated with methylated bovine serum albumn (mBSA) reacted with a serum sample, then indicated by a well-known fluorescent anti-globulin method. Further, by changing the antigen from DNA to a thymic extract, claims are drawn to detection of antibodies to extractable nuclear antigen (ENA). These ENA are not identified per se and the tests for SLE as described could not be used to detect MCTD or vise versa.
U.S. Pat. No. 3,897,212 to S. A. Leon, et al. July 29, 1975 discloses a direct radioimmunoassay for antibodies to DNA in SLE patients, by reacting a serum sample with radioactively labeled DNA and measuring the amount of radioactivity in a precipitate of the DNA and test serum.
U.S. Pat. No. 3,997,657 to B. F. Dziobkowski Dec. 14, 1976 discloses a dry slide technique for the detection of human antinuclear factor. The method involves reacting the test serum with a thymus cell extract, which had been previously affixed to a glass slide, followed by an immunofluorescent assay. No attempt was made to identify any components of the extract.
U.S. Pat. No. 4,314,987 to R. I. Morris et al. Feb. 9, 1982 discloses a method of diagnosing rheumatological diseases based upon patterns of fluorescence (i.e. homogeneous rim, speckled, nucleolar, etc.) of antinuclear antibodies, followed by testing for anti-DNA or anti-ENA antibodies. Although an ability to distinguish between SLE and MCTD is claimed, the patent essentially provides a strategy for performing and interpreting already existing tests and as such is limited in accuracy by the tests themselves. Further no data are presented to allow one skilled in the art to evaluate the efficacy of such an approach.
Contrary to teaching of U.S. Pat. No. 4,314,987 wherein nucleolar patterns of fluorescence were considered to be indefinite, the very existence of such nucleolar fluorescence (Pinnas, J L et al. J. of Immunol. 111(4):996-1004 (1973)) provided the impetus for the research upon which the subject invention is based.